Modified envelope glycoproteins for retroviridae viral vector pseudotyping and process for obtaining it

Pending Publication Date: 2022-06-30
INST DE BIOLOGIA EXPERIMENTAL E TECH IBET
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of a highly pathogenic human virus for cargo delivery in therapeutic applications raises serious biosafety concerns.
With this system, good titers were easily achieved, but the poor safety level could not be accepted for a human and potentially lethal pathogen.
These packaging systems are not, however, easily available for the research community.
The natural tropism of HIV-1 envelope glycoprotein is

Method used

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  • Modified envelope glycoproteins for retroviridae viral vector pseudotyping and process for obtaining it
  • Modified envelope glycoproteins for retroviridae viral vector pseudotyping and process for obtaining it
  • Modified envelope glycoproteins for retroviridae viral vector pseudotyping and process for obtaining it

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example 1

or Lentiviral Vectors Production and Titration

[0100]For transient production of lentiviral vectors, the third generation lentiviral packaging system and the transfection procedure as described in Tomás et al. (2013 and 2018) were used (Tomás, H. A., Rodrigues, A. F., Alves, P. M. Coroadinha, A. S. Lentiviral Gene Therapy Vectors: Challenges and Future Directions. Gene Therapy—Tools and Potential Applications (ed. Martin, F.). InTech (2013) pp. 287-317), (Tomás HA, Rodrigues A F, Carrondo M J T, Coroadinha A S. LentiPro26: novel stable cell lines for constitutive lentiviral vector production. Sci Rep. 8(1):5271. 2018).

[0101]The transfection procedure was conducted using PEI. HEK 293T cells were seeded at 5×104 cells / cm2 in 25 cm2 t-flask 24 h prior to transfection. A total of 4.65 μg of plasmid DNA per million cells was used for the transfection of one t-flask: 1 μg of pMDLg / pRRE or its variants (T26S and D25N) and 0.25 μg of pRSV-Rev (providing the packaging functions), 2.5 μg of pR...

example 2

on of the Modified Envelope Glycoproteins for Viral Vector Pseudotyping

[0112]Gammaretrovirus envelope glycoproteins, unlike VSV-G, undergo proteolytic processing during virion assembly mediated by the retroviral protease. A short sequence—R-peptide—is cleaved from the cytoplasmic tail, as described by Tedbury, P. R. & Freed, E. O. (Tedbury, P. R. & Freed, E. O. The cytoplasmic tail of retroviral envelope glycoproteins. Prog. Mol. Biol. Transl. Sci. 129, 253-84. 2015). This cleavage is required for virus entry, since it activates the fusogenic activity of the envelope glycoprotein.

[0113]The R-peptide cleavage site in the original envelope glycoproteins is specifically recognized by the retroviral protease. The efficiency of cleavage is dependent on both the sequence of cleavage and the protease used (i.e. its virus family origin and introduced mutations). Enhanced cleavage is expected when (i) homologous cleavage sequences, in relation to the viral protease, are used and (ii) highly ...

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Abstract

The present invention describes the development of a modified envelope glycoproteins to pseudo-type viruses of the retroviridae family. These are derived from Murine leukaemia virus amphotropic, Gibbon Ape leukaemia virus and feline endogenous virus envelopes.
The improved envelope glycoproteins contain, among other modifications, newly introduced alternative cleavable sequences.
The viral vectors pseudo-typed with these modified envelopes may be suitably employed for cargo delivery such as in gene and cell therapy applications, for the ex vivo and in vivo delivery of gene(s), protein(s), or molecule(s) of interest to a variety of target cells.

Description

TECHNICAL DOMAIN OF THE INVENTION[0001]The present invention relates to the development of modified envelope glycoproteins to pseudo-type viruses of the retroviridae family. These are derived from Murine leukaemia virus amphotropic, Gibbon Ape leukaemia virus and feline endogenous virus envelopes. The improved envelope glycoproteins contain, among other modifications, newly introduced alternative cleavable sequences.[0002]The viral vectors pseudo-typed with these modified envelopes may be suitably employed for cargo delivery such as in gene and cell therapy applications, for the ex vivo and in vivo delivery of gene(s), protein(s), or molecule(s) of interest to a variety of target cells.[0003]The modified envelope glycoproteins to pseudo-type viruses can be utilised using transient co-transfection of plasmids system or to develop stable cell lines producing recombinant viruses.[0004]Therefore, the present invention is in the area of genetic engineering, diagnose, pharmaceutic and med...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/86
CPCC12N7/00C12N15/86C12N2740/15043C12N2740/13052C12N2740/15051C12N2740/13022C07K14/005C12N2740/13045C12N2740/15045C12N2830/42C12N2830/48C12N2830/50C12N2740/16043C12N2740/16045C12N2740/16052C07K2319/50C07K2319/03
Inventor ANTUNES TOMÁS, HÉLIOFERNANDES MESTRE, DANIEL ALEXANDREDE ALBUQUERQUE FERREIRA RODRIGUES, ANA FILIPADE SOUSA VALENTE COROADINHA, ANA SOFIA
Owner INST DE BIOLOGIA EXPERIMENTAL E TECH IBET
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